Method for treating a demyelinating condition

ABSTRACT

Methods for treating a demyelinating condition in a subject in need of treatment are provided. In some aspects the methods encompass administering to the subject an amount of a Ca 2+ -channel blocker effective to treat the demyelinating condition. In other aspects, the methods encompass administering to the subject an amount of a glutamate inhibitor effective to treat the demyelinating condition. In additional aspects, the methods encompass administering to the subject a Ca 2+ -channel blocker in combination with a glutamate inhibitor, in amounts effective to treat the demyelinating condition. In still other aspects, the methods encompass administering to the subject a Ca 2+ -channel blocker in combination with a hypertensive agent, in amounts effective to treat the demyelinating condition. Also provided are pharmaceutical compositions having a Ca 2+ -channel blocker, a glutamate inhibitor, and a pharmaceutically-acceptable carrier. Additionally, pharmaceutical compositions having a Ca 2+ -channel blocker, a hypertensive agent, and a pharmaceutically-acceptable carrier are provided.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This is a continuation-in-part of U.S. patent application Ser.No. 09/678,686, filed Oct. 3, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The U.S. Government has a paid-up license in this invention andthe right in limited circumstances to require the patent owner tolicense others on reasonable terms as provided for by the terms ofNS41056 and NS07098 awarded by the National Institutes of Health.

BACKGROUND OF THE INVENTION

[0003] (1) Field of the Invention

[0004] The present invention generally relates to treatments ofdemyelinating conditions. More specifically, the invention relates tothe use of calcium channel blockers for treating demyelinatingconditions, especially multiple sclerosis.

[0005] (2) Description of the Related Art

[0006] References Cited

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[0061] PCT patent publication WO 92/07564.

[0062] Demyelination is a feature of many neurologic disorders.Demyelinating conditions are manifested in loss of myelin—the multipledense layers of lipids and protein which cover many nerve fibers. Theselayers are provided by oligodendroglia in the central nervous system(CNS), and Schwann cells in the peripheral nervous system. In patientswith demyelinating conditions, demyelination may be irreversible; it isusually accompanied or followed by axonal degeneration, and often bycellular degeneration. Demyelination can occur as a result of neuronaldamage or damage to the myelin itself—whether due to aberrant immuneresponses, local injury, ischemia, metabolic disorders, toxic agents, orviral infections (Prineas and McDonald, 1997; Beers and Berkow, 1999).

[0063] Central demyelination (demyelination of the CNS) occurs inseveral conditions, often of uncertain etiology, that have come to beknown as the primary demyelinating diseases. Of these, multiplesclerosis is the most prevalent. Other primary demyelinating diseasesinclude adrenoleukodystrophy, adrenomyeloneuropathy, AIDS-vacuolarmyelopathy, HTLV-associated myelopathy, Leber's hereditary opticatrophy, progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, and tropical spastic paraparesis. In addition, thereare acute conditions in which demyclination can occur in the CNS, e.g.,acute disseminated encephalomyelitis and acute viral encephalitis.Furthermore, acute transverse myelitis, a syndrome in which an acutespinal cord transection of unknown cause affects both gray and whitematter in one or more adjacent thoracic segments, can also result indemyelination. Finally, there are animal models which mimic features ofhuman demyelinating diseases (Beers and Berkow, 1999). Examples includeexperimental autoimmune neuritis, demyelination induced by Theiler'svirus, and experimental autoimmune encephalomyelitis (EAE)—an autoimmunedisease which is experimentally induced in a variety of species andwhich resembles multiple sclerosis in its clinical and neuropathologicalaspects (Gold et al., 2000; Njenga and Rodriguez, 1996).

[0064] Multiple sclerosis (MS) is the most prevalent demyclinatingcondition. In Europe and North America, an average of 40-100 people outof every 100,000 have MS. The disease affects approximately 250,000people in the United States alone. MS is a chronic, devastatingneurological disease that affects mostly young adults. The pathogenesisof MS is a complex process that leads to destruction of myelin andoligodendroglia, as well as axonal damage, in the brain and spinal cord(Prineas and McDonald, 1997; Trapp et al., 1998). Histopathologically,MS is characterized by inflammation, plaques of demyelinationinfiltrating cells in the CNS tissue, loss of oligodendroglia, and focalaxonal injury (Prineas and McDonald, 1997). The disease is thought toresult from aberrant immune responses to myelin, and possiblynon-myelin, self-antigens (Bar-Or et al., 1999; Hartung, 1995).Clinically, MS may follow a relapsing-remitting, or it may take achronically progressive course with increasing physical disability (Goldet al., 2000). Typically, the symptoms of MS include lack ofco-ordination, paresthesias, speech and visual disturbances, andweakness (Beers and Berkow, 1999).

[0065] Current treatments for the various demyelinating conditions areoften expensive, symptomatic, and only partially effective, and maycause undesirable secondary effects. Corticosteroids (oral prednisone at60-100 mg/day, tapered over 2-3 weeks, or intravenous methylprednisoloneat 500-1000 mg/day, for 3-5 days) represent the main form of therapy forMS. While these may shorten the symptomatic period during attacks, theymay not affect eventual long-term disability. Long-term corticosteroidtreatment is rarely justified, and can cause numerous medicalcomplications, including osteoporosis, ulcers, and diabetes (Id.).

[0066] Immunomodulatory therapy with recombinant human interferon-β(Betaseron and Avonex) and with co-polymer (Copaxon) slightly reducesthe frequency of relapses in MS, and may help delay eventual disability(Id.). Both forms of interferon-β and co-polymer are currently used astreatment modalities for MS, but all are exceedingly expensive.Immunosuppressive drugs (azathioprine, cladribine, cyclophosphamide, andmethotrexate) are used for more severe progressive forms. However, theyare not uniformly beneficial, and have significant toxic side-effects.Several drugs (e.g., baclofen at 30-60 mg/day in divided doses) mayreduce spasticity by inhibiting the spinal cord reflexes. Cautious andjudicious use is required, though, because the drug-induced reduction inspasticity in MS patients often exacerbates weakness, thereby furtherincapacitating the patient (Id.).

[0067] Similarly, current treatment for adrenoleukodystrophy, anotherdevastating demyelinating disease, is relatively ineffective. Symptomsof adrenoleukodystrophy may include cortical blindness, corticospinaltract dysfunction, mental deterioration, and spasticity. Therapy tocontrol the course of adrenoleukodystrophy may include bone marrowtransplantation and dietary treatment (DiBiase et al., 1999), butinexorable neurological deterioration invariably occurs, ultimatelyleading to death (Krivit et al., 1999). Some progress has been realizedin the treatment of animals with EAE and experimental autoimmuneneuritis, by using glial cell transplants and growth factors, and byinhibiting adhesion molecules, autoantibodies, and cytokines (Njenga andRodriguez, 1996). However, none of these treatments has been shown to bebeneficial in humans, and some require extensive neurosurgicalintervention. Thus, it is clear from the foregoing that there exists aneed for more effective, and less expensive and invasive, methods totreat the varied array of demyelinating conditions, without producingundesirable secondary effects.

[0068] Calcium-channel blockers are a class of pharmacological agentswhich inhibit the transmembrane flux of calcium (Ca²⁺) ions into cells,particularly vascular smooth muscle cells and cardiac muscle cells. Theyhave been indicated for the treatment of angina, arrhythmias, atrialfibrillation, hypertension, and paroxysmal supraventricular tachycardia(Physicians' Desk Reference, 2000). Amlodipine, a potent Ca²⁺-channelblocker, is a long-acting dihydropyridine calcium antagonist (calciumion antagonist or slow-channel blocker). Amlodipine selectively inhibitsCa²⁺-ion influx across cell membranes, with a greater effect on vascularsmooth muscle cells than on cardiac muscle cells. In particular,amlodipine is a peripheral arterial vasodilator that acts directly onvascular smooth muscle to cause a reduction in peripheral vascularresistance and a reduction in blood pressure. Amlodipine has beendemonstrated to be effective in treating chronic stable angina,vasospastic angina, and hypertension (Id.), and it may also haveneuroprotective activity (Mason et al., 1999). Other Ca²⁺-channelblockers include bepridil, nitrendipine, diltiazem, felodipine,flunarizine, isradipine, mibefradil, nicardipine, nifedipine,nimodipine, nisoldipine, nivaldipine, and verapamil (Physicians' DeskReference, 2000).

[0069] There has been a previous suggestion that calcium-channelblockers could be effective for demyelinating conditions. PCT patentpublication WO 92/07564, to the Wellcome foundation (“Wellcome”), claimssuch a use, particularly using nimodipine. However, the skilled artisanwould not consider Wellcome to be an enabling disclosure because thecell culture experiment disclosed therein would not be understood toestablish the effectiveness of calcium-channel blockers fordemyelinating conditions, especially inflammatory demyelinatingconditions such as MS.

SUMMARY OF THE INVENTION

[0070] The present invention is predicated on the discovery thatcalcium-channel blockers ameliorate the clinical impairment of ademyelinating condition, experimental autoimmune encephalomyelitis(EAE), which is commonly used as a model of multiple sclerosis (MS). Onthe basis of these findings, the present invention provides methods fortreating a demyclinating condition in a subject in need of treatment.The methods comprise administering to the subject an amount of acalcium-channel blocker effective to treat the demyelinating condition.

[0071] The invention is also directed to additional methods for treatinga demyelinating condition in a subject in need of treatment. The methodscomprise administering to the subject a glutamate inhibitor, in amountseffective to treat the demyelinating condition.

[0072] In related embodiments, the present invention is directed toother methods for treating a demyelinating condition in a subject inneed of treatment. These methods comprise administering to the subject acalcium-channel blocker in combination with a glutamate inhibitor, inamounts effective to treat the demyelinating condition.

[0073] The invention is additionally directed to further methods fortreating a demyelinating condition in a subject in need of treatment.These methods comprise administering to the subject a calcium-channelblocker in combination with a hypertensive agent, in amounts effectiveto treat the demyelinating condition.

[0074] In further related embodiments, the invention is directed toadditional methods for treating a demyelinating condition in a subjectin need of treatment. These methods comprise administering to thesubject a combination of a calcium-channel blocker, a glutamateinhibitor and a hypertensive agent.

[0075] In additional embodiments, the invention is directed topharmaceutical compositions comprising a calcium-channel blocker, aglutamate inhibitor, and a pharmaceutically-acceptable carrier.

[0076] Additionally, the invention is directed to pharmaceuticalcompositions comprising a calcium-channel blocker, a hypertensive agent,and a pharmaceutically-acceptable carrier.

[0077] The invention is also directed to a pharmaceutical compositioncomprising a calcium-channel blocker, a glutamate inhibitor, ahypertensive agent, and a pharmaceutically-acceptable carrier.

[0078] Additional objects of the present invention will be apparent inview of the description which follows.

BRIEF DESCRIPTION OF THE FIGURES

[0079]FIG. 1 illustrates the clinical course of adoptive-transfer EAEand the effect of treatment with amlodipine. SJL mice were injected with3×10⁷ MBP-activated cells. Starting from Day 5 post-immunization, micewere treated with amlodipine (30 μg as one daily subcutaneous injection)or vehicle (PBS), until Day 13 (Day 9 of treatment). Data representmean±s.e.m. differences, at respective time-points, between thevehicle-treated group and the amlodipine-treated group. *=p<0.05;**=p<0.01 (students' unpaired, two-tailed t-test); n=8 per group.

[0080]FIG. 2 is a graph summarizing experiments that establish thatbepridil administration beginning at day 3 after disease inductionattenuates adoptive-transfer EAE. Mice receiving bepridil (3 mg/kg; opencircles) starting at day 3 post-treatment showed a significantly reducedneurological disability compared with mice treated with correspondingvehicle (closed circles). The significance of differences in diseasescore between treatment groups was assessed by analysis of variancefollowed by multiple comparisons.

[0081]FIG. 3 is a graph summarizing experiments that establish thatbepridil administration beginning on the same day of disease inductionattenuates adoptive-transfer EAE. Mice receiving bepridil (3 mg/kg; opencircles) starting at the day of treatment showed a significantly reducedneurological disability compared with mice treated with correspondingvehicle (closed circles). The significance of differences in diseasescore between treatment groups was assessed by analysis of variancefollowed by multiple comparisons.

[0082]FIG. 4 is a graph summarizing experiments that establish thatnitrendipine administration on the same day of disease inductionattenuates adoptive-transfer EAE. Mice receiving nitrendipine (2.5mg/kg; open circles) starting at the day of treatment showed asignificantly reduced neurological disability compared with mice treatedwith corresponding vehicle (closed circles). The significance ofdifferences in disease score between treatment groups was assessed byanalysis of variance followed by multiple comparisons.

[0083]FIG. 5 shows micrographs of vehicle-(Panel A) and bepridil-(PanelB) treated mice with adoptive transfer EAE. Vehicle treated mice withadoptive transfer EAE (Panel A) have widespread regions which areinfiltrated with monocytes and are devoid of axons. These regions areflanked by areas that contain axons with abnormal myelin (asterisk). Onthe right, a region with many intact axons. Bepridil treated mice withadoptive transfer EAE (Panel B) have fewer infiltrating cells with onlysporadic axonal abnormalities (asterisk).

DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention is based on the discovery, outlined in theExamples, that calcium-channel blockers are effective in reducing theeffects of demyelinating conditions, in particular multiple sclerosis(MS). This discovery, and other discoveries disclosed in the Examples,demonstrate that various methods and compositions are useful fortreating demyelinating conditions.

[0085] Thus, in some embodiments, the invention is directed to methodsfor treating a demyelinating condition in a subject in need oftreatment. The methods comprise administering to the subject an amountof a formulation of a calcium-channel blocker (Ca²⁺-channel blocker)effective to treat the demyelinating condition in the subject. As usedherein, the term “demyelinating condition” refers to a disease,disorder, or condition characterized by loss of myelin. Examplesinclude, without limitation, acute disseminated encephalomyelitis, acutetransverse myelitis, acute viral encephalitis, adrenoleukodystrophy,adrenomyeloneuropathy, AIDS-vacuolar myclopathy, experimental autoimmuneencephalomyelitis (EAE), experimental autoimmune neuritis,HTLV-associated myelopathy, Leber's hereditary optic atrophy, multiplesclerosis (MS), progressive multifocal leukoencephalopathy, subacutesclerosing panencephalitis, and tropical spastic paraparesis.Preferably, the demyelinating condition is MS.

[0086] Additionally, as used herein, the term “calcium-channel blocker”or “Ca²⁺-channel blocker” refers to any one of a class ofpharmacological agents, also known as calcium antagonists, which inhibitthe transmembrane flux of calcium (Ca²⁺) ions. As used herein, the term“agent” includes a protein, polypeptide, peptide, nucleic acid(including DNA or RNA), antibody, molecule, compound, antibiotic, drugand any combinations thereof. Numerous calcium-channel blockers areknown in the art. Preferred examples of calcium-channel blockers aresmall molecular weight (i.e., less than about 1000 molecular weight)aromatic compounds that are known as calcium channel blockers, forexample amlodipine, bepridil, nitrendipine, diltiazem, felodipine,flunarizine, isradipine, mibefradil, nicardipine, nifedipine,nimodipine, nisoldipine, nivaldipine, and verapamil. As used herein,these compounds include any non-covalent chemical derivatives of theactive chemical, such as a salt, free base, hydrate, hydrochloride, etc.

[0087] As used herein, “formulation” is a preparation of an agent orcombination of agents. The combination of agents can be preparedseparately or mixed together.

[0088] Since the therapeutic effectiveness of these methods is based onthe ability of the agent to inhibit the transmembrane flux of calciumions, any calcium-channel blocker would be expected to provide benefit.For example, the Examples establish that three calcium-channel blockers,amlodipine, bepridil and nitrendipine, are effective. Two of thoseagents, amlodipine and nitrendipine, are dihydropyridine-typecalcium-channel blockers, but bepridil is not chemically related todihydropyridine or any other type of calcium-channel blocker. Althoughany calcium-channel blocker would be expected to be effective againstdemyelinating conditions, the effectiveness of any particularcalcium-channel blocker could be easily tested, for example by using themethods provided in the Examples.

[0089] In these embodiments, the subject may be any mammal but ispreferably a human. Preferably, the calcium-channel blocker isamlodipine, bepridil or nitrendipine.

[0090] As used herein, “amlodipine” is3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate.Synthetic amlodipine is commercially available, and can be obtained fromPfizer Inc. (New York, N.Y.). Norvasc® is the besylate salt ofamlodipine. Norvasc® tablets are formulated as white tablets, equivalentto 2.5, 5; and 10 mg of amlodipine, for oral administration. Amlodipineis an affordable compound; moreover, it provides a novel approach totreating demyelinating conditions based on pathophysiologic mechanisms(Pitt et al., 2000). As with other calcium-channel blockers, though,amlodipine should be used with caution when treating subjects with heartfailure (Physicians' Desk Reference, 2000).

[0091] Nitrendipine is known in the art as ethylmethyl-1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylate.It is currently not known by any brand name.

[0092] Bepridil is known in the art as[(2-methoxypropoxy)methyl]-N-phenyl-N-(phenylmethyl)-I-pyrrolidineethanamine.Bepridil is sold commercially as Vascor® (Ortho-McNeil Pharmaceutical,Inc., Raritan, N.J.), which is the monohydrochloride monohydrate form ofthe chemical.

[0093] In these methods, the calcium-channel blocker is administered toa subject having a demyelinating condition in an amount which iseffective to treat the demyelinating condition in the subject. As usedherein, the phrase “effective to treat the demyelinating condition”means effective to ameliorate or minimize the clinical impairment orsymptoms of the demyelinating condition. For example, where thedemyelinating condition is MS, the amount of calcium-channel blockereffective to treat the demyelinating condition is that which canameliorate or minimize the symptoms of MS, including lack ofco-ordination, paresthesias, speech and visual disturbances, andweakness. The amount of calcium-channel blocker effective to treat ademyelinating condition in a subject will vary depending on thecalcium-channel blocker which is used. For example, the amount ofamlodipine may range from about 5 mg/day to about 35 mg/day. Appropriateamounts of other calcium-channel blockers effective to treat ademyelinating condition in a subject can be readily determined by theskilled artisan without undue experimentation.

[0094] According to the method of the present invention, thecalcium-channel blocker may be administered to a human or animal subjectby known procedures, including, but not limited to, oral administration,parenteral administration, transdermal administration, andadministration through an osmotic mini-pump. Preferably, thecalcium-channel blocker is administered orally.

[0095] For oral administration, the formulation of the calcium-channelblocker may be presented as capsules, tablets, powders, granules, or asa suspension. The formulation may have conventional additives, such aslactose, mannitol, corn starch, or potato starch. The formulation alsomay be presented with binders, such as crystalline cellulose, cellulosederivatives, acacia, corn starch, or gelatins. Additionally, theformulation may be presented with disintegrators, such as corn starch,potato starch, or sodium carboxymethylcellulose. The formulation alsomay be presented with dibasic calcium phosphate anhydrous or sodiumstarch glycolate. Finally, the formulation may be presented withlubricants, such as talc or magnesium stearate.

[0096] For parenteral administration, the calcium-channel blocker may becombined with a sterile aqueous solution which is preferably isotonicwith the blood of the subject. Such a formulation may be prepared bydissolving a solid active ingredient in water containingphysiologically-compatible substances, such as sodium chloride, glycine,and the like, and having a buffered pH compatible with physiologicalconditions, so as to produce an aqueous solution, then rendering saidsolution sterile. The formulations may be present in unit or multi-dosecontainers, such as sealed ampoules or vials. The formulation may bedelivered by any mode of injection, including, without limitation,epifascial, intracapsular, intracutaneous, intramuscular, intraorbital,intraspinal, intrasternal, intravascular, intravenous, parenchymatous,or subcutaneous.

[0097] For transdermal administration, the calcium-channel blocker maybe combined with skin penetration enhancers, such as propylene glycol,polyethylene glycol, isopropanol, ethanol, oleic acid,N-methylpyrrolidone, and the like, which increase the permeability ofthe skin to the calcium-channel blocker, and permit the calcium-channelblocker to penetrate through the skin and into the bloodstream. Thecalcium-channel blocker/enhancer compositions also may be furthercombined with a polymeric substance, such as ethylcellulose,hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone,and the like, to provide the composition in gel form, which may bedissolved in solvent such as methylene chloride, evaporated to thedesired viscosity, and then applied to backing material to provide apatch.

[0098] The calcium-channel blocker of the present invention also may bereleased or delivered from an osmotic mini-pump. The release rate froman elementary osmotic mini-pump may be modulated with a microporous,fast-response gel disposed in the release orifice. An osmotic mini-pumpwould be useful for controlling release of, or targeting delivery of, acalcium-channel blocker, particularly a short-acting calcium-channelblocker.

[0099] The inventors have also discovered that blocking glutamateexcitotoxicity mediated by AMPA/Kainate receptors are effective inreducing the effects of demyelinating conditions, in particular multiplesclerosis (MS). See Pitt et al., 2000. Accordingly, some embodiments ofthe present invention are directed to methods for treating ademyelinating condition in a subject in need of treatment. The methodscomprise administering to the subject a formulation of a glutamateinhibitor, in amounts effective to treat the demyelinating condition. Aswith the calcium-channel blocker embodiments, the subject is preferablya human and the demyelinating condition is preferably MS.

[0100] As used herein, the term “glutamate inhibitor” refers to any of aclass of pharmacological agents which prevent the binding and/or actionof glutamate (or glutamatergic agonists) at ionotropic glutamatereceptors, resulting in reduced or completely blocked ion-conductance ofsuch receptors. Examples of appropriate glutamate inhibitors include,without limitation, carbidopa, levodopa, sodium-channel blockers, andNBQX(1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzoquinoxaline-7-sulfonamide).The methods of administration of the calcium-channel blockers discussedabove would generally be expected to be useful for administration ofglutamate inhibitors.

[0101] The present invention is also directed to a method for treating ademyclinating condition in a subject in need of treatment, comprisingadministering to the subject a formulation of a calcium-channel blockerin combination with a glutamate inhibitor, in amounts effective to treatthe demyelinating condition. The demyelinating condition may be any ofthose described above. The calcium-channel blocker may also be any ofthose described above.

[0102] In the method of the present invention, administration of acalcium-channel blocker “in combination with” another compound, in thiscase a glutamate inhibitor, refers to co-administration of the twocompounds. Co-administration may occur concurrently, sequentially, oralternately. Concurrent co-administration refers to administration ofboth a calcium-channel blocker and a glutamate inhibitor at essentiallythe same time. For concurrent co-administration, the courses oftreatment with a calcium-channel blocker and with a glutamate inhibitormay be run simultaneously. For example, a single, combined formulation,containing both an amount of a calcium-channel blocker and an amount ofa glutamate inhibitor in physical association with one another, may beadministered to the subject. The single, combined formulation mayconsist of an oral formulation, containing amounts of both acalcium-channel blocker and a glutamate inhibitor, which may be orallyadministered to the subject, or a liquid mixture, containing amounts ofboth a calcium-channel blocker and a glutamate inhibitor, which may beinjected into the subject.

[0103] It is also within the confines of the present invention that acalcium-channel blocker and a glutamate inhibitor may be administeredconcurrently to a subject, in separate, individual formulations.Accordingly, the method of the present invention is not limited toconcurrent co-administration of a calcium-channel blocker and aglutamate inhibitor in physical association with one another.

[0104] In the method of the present invention, a calcium-channel blockerand a glutamate inhibitor also may be co-administered to a subject in aformulation comprising separate, individual preparations that are spacedout over a period of time, so as to obtain the maximum efficacy of thecombination. Administration of each agent may range in duration, from abrief, rapid administration to a continuous perfusion. When spaced outover a period of time, co-administration of a calcium-channel blockerand a glutamate inhibitor may be sequential or alternate. For sequentialco-administration, one of the agents is separately administered,followed by the other. For example, a full course of treatment with acalcium-channel blocker may be completed, and then may be followed by afull course of treatment with a glutamate inhibitor. Alternatively, forsequential co-administration, a full course of treatment with aglutamate inhibitor may be completed, then followed by a full course oftreatment with a calcium-channel blocker. For alternateco-administration, partial courses of treatment with a calcium-channelblocker may be alternated with partial courses of treatment with aglutamate inhibitor, until a full treatment of each agent has beenadministered.

[0105] The agents of the present invention (i.e., a calcium-channelblocker and a glutamate inhibitor, either in separate, individualformulations, or in a single, combined formulation) may be administeredto a human or animal subject by any known procedures, including all ofthe above-described methods. Preferably, however, the calcium-channelblocker and the glutamate inhibitor are co-administered orally.

[0106] In the method of the present invention, a calcium-channel blockerand a glutamate inhibitor are co-administered in amounts effective totreat the demyelinating condition in the subject. As described above,this means that an amount of calcium-channel blocker in combination withan amount of glutamate inhibitor is effective to ameliorate or minimizethe clinical impairment or symptoms of the demyelinating condition.Appropriate amounts of a calcium-channel blocker and a glutamateinhibitor effective to treat a demyelinating condition in a subject canbe readily determined by the skilled artisan. A calcium-channel blockerand a glutamate inhibitor may be co-administered to a subject in orderto achieve a synergistic effect in the treatment of a demyelinatingcondition.

[0107] Since it is well known that calcium-channel blockers reduce bloodpressure, the skilled artisan would understand that it may be necessaryto administer a hypertensive agent along with the calcium-channelblocker, in order to reduce the possible deleterious hypotensive effectof the calcium-channel blocker. Thus, in some embodiments, the presentinvention provides additional methods for treating a demyelinatingcondition in a subject in need of treatment. These methods compriseadministering to the subject a calcium-channel blocker in combinationwith a hypertensive agent, in amounts effective to treat thedemyelinating condition and to counter the hypotensive effect of thecalcium-channel blocker. The demyelinating condition may be any of thosedescribed above. The calcium-channel blocker may also be any of thosedescribed above. Additionally, as used herein, the term “hypertensiveagent” refers to any of a class of pharmacological agents which increaseblood pressure. As described above, an “agent” includes a protein,polypeptide, peptide, nucleic acid (including DNA or RNA), antibody,molecule, compound, antibiotic, drug, and any combinations thereof.However, in preferred embodiments the hypertensive agent is any smallmolecule (i.e., less than 1000 MW) known to have a hypertensive effect.Examples of appropriate hypertensive agents include, without limitation,phenylephrine (i.e., hydroxy-α-[(methylamino)methyl]benzyl alcohol)(particularly phenylephrine that has been carefully titrated) and sodiumchloride (NaCl).

[0108] In the method of the present invention, administration of acalcium-channel blocker “in combination with” a hypertensive agentrefers to co-administration of a formulation of the two agents. Asdescribed above, co-administration may occur concurrently, sequentially,or alternately. A calcium-channel blocker and a hypertensive agent maybe co-administered by any of the above-described methods, and in any ofthe above-described formulations. For example, for concurrentco-administration, as described above, the courses of treatment with acalcium-channel blocker and with a hypertensive agent may be runsimultaneously, in a single, combined formulation containing both anamount of a calcium-channel blocker and an amount of a hypertensiveagent in physical association with one another. Alternatively, asdescribed above, an amount of a calcium-channel blocker and an amount ofa hypertensive agent may be administered concurrently to a subject, inseparate, individual preparations. Accordingly, the method of thepresent invention is not limited to concurrent co-administration of acalcium-channel blocker and a hypertensive agent in physical associationwith one another.

[0109] In the method of the present invention, a calcium-channel blockerand a hypertensive agent also may be co-administered to a subject inseparate, individual preparations that are spaced out over a period oftime, so as to obtain the maximum efficacy of the combination.Administration of each agent may range in duration, from a brief, rapidadministration to a continuous perfusion. When spaced out over a periodof time, co-administration of a calcium-channel blocker and ahypertensive agent may be sequential or alternate, as described above.

[0110] The agents of the present invention (i.e., a calcium-channelblocker and a hypertensive agent, either in separate, individualpreparations, or in a single, combined formulation) may be administeredto a human or animal subject by any known procedures, including all ofthe above-described methods. Preferably, however, the calcium-channelblocker and the hypertensive agent are co-administered orally.

[0111] In the method of the present invention, a calcium-channel blockerand a hypertensive agent are co-administered in amounts effective totreat the demyelinating condition in the subject. As described above,this means that an amount of calcium-channel blocker in combination withan amount of hypertensive agent is effective to ameliorate or minimizethe clinical impairment or symptoms of the demyelinating condition.Appropriate amounts of a calcium-channel blocker and a hypertensiveagent effective to treat a demyelinating condition in a subject can bereadily determined by the skilled artisan. A calcium-channel blocker anda hypertensive agent may be co-administered to a subject in order toachieve a synergistic effect in the treatment of a demyelinatingcondition.

[0112] Based on the discussion above pertaining to combinations ofcalcium-channel blockers with glutamate inhibitors or hypotensiveagents, it would also be understood that combinations of these threeagents can be used to treat demyelinating conditions. Thus, theinvention is directed to additional methods for treating a demyelinatingcondition in a subject in need of treatment. These methods compriseadministering to the subject an amount of an agent effective to treatthe demyelinating condition, where the agent is a combination ofcalcium-channel blocker, a glutamate inhibitor and a hypertensive agent.

[0113] It is within the confines of the present invention that theformulations of a calcium-channel blocker and a glutamate inhibitor, acalcium-channel blocker and a hypertensive agent, or a calcium-channelblocker, glutamate inhibitor and a hypertensive agent (whetherindividual or combined for any of these formulations) may be furtherassociated with a pharmaceutically-acceptable carrier, therebycomprising a pharmaceutical composition. Accordingly, the presentinvention is also directed to pharmaceutical compositions comprising acalcium-channel blocker, a glutamate inhibitor, and apharmaceutically-acceptable carrier; in other embodiments thepharmaceutical composition is a calcium-channel blocker, a hypertensiveagent, and a pharmaceutically-acceptable carrier; in still otherembodiments the pharmaceutical composition is a calcium-channel blocker,a glutamate inhibitor, a hypertensive agent and apharmaceutically-acceptable carrier. Any of these pharmaceuticalcompositions would be useful for treating a demyelinating condition in asubject in need of treatment. Where the pharmaceutical composition isadministered to a subject to treat a demyelinating condition, thecalcium-channel blocker and a glutamate inhibitor are provided inamounts which are effective to treat the demyelinating condition.

[0114] The pharmaceutically-acceptable carrier of the present inventionmust be “acceptable” in the sense of being compatible with the otheringredients of the composition, and not deleterious to the recipientthereof. Examples of acceptable pharmaceutical carriers includecarboxymethylcellulose, crystalline cellulose, glycerin, gum arabic,lactose, magnesium stearate, methyl cellulose, powders, saline, sodiumalginate, sucrose, starch, talc, and water, among others. Formulationsof the pharmaceutical composition may conveniently be presented in unitdosage.

[0115] The formulations of the present invention may be prepared bymethods well-known in the pharmaceutical art. For example, the activecompound may be brought into association with a carrier or diluent, as asuspension or solution. Optionally, one or more accessory ingredients(e.g., buffers, flavoring agents, surface active agents, and the like)also may be added. The choice of carrier will depend upon the route ofadministration. The pharmaceutical composition would be useful foradministering the calcium-channel blocker and the glutamate inhibitor ofthe present invention (either in separate, individual formulations, orin a single, combined formulation) to a subject to treat a demyelinatingcondition. The agents are provided in amounts that are effective totreat a demyelinating condition in the subject. These amounts may bereadily determined by the skilled artisan.

[0116] Preferred embodiments of the invention are described in thefollowing examples. Other embodiments within the scope of the claimsherein will be apparent to one skilled in the art from consideration ofthe specification or practice of the invention as disclosed herein. Itis intended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE 1 Treatment of Experimental Autoimmune Encephalomyelitis withAmlodipine

[0117] Materials and Methods

[0118] Induction of Experimental Autoimmune Encephalomyelitis (EAE).Adoptive-transfer EAE was induced in female SJL mice as described(Cannella et al., 1998). In brief, lymph node cells were obtained 10days after myelin basic protein/Complete Freund's Adjuvant immunization.Cells were cultured for 4 days with 50 μg/ml of myelin basic protein,then 3×10⁷ cells/mouse were injected into syngeneic mice via tail veins.Onset of disease occurred after 7-9 days. Animals were graded accordingto a standard clinical index (0-5). Five days after immunization,animals began treatments with one daily injection of 200 μl of vehicle(PBS), or 30 μg of amlodipine in 200 μl of PBS.

[0119] Neuropathology. At selected time-points, mice from the controland treated groups were perfused with PBS or glutaraldehyde, and the CNSwas prepared for frozen or 1-μm epoxy sections, respectively. Epoxysections were stained with toluidine blue, and examined by lightmicroscopy. Frozen sections were used for immunohistochemistry toevaluate damage to oligodendrocytes and neurons.

[0120] Results and Discussion

[0121] Multiple sclerosis (MS) is characterized by destruction of myelinand oligodendrocytes in the CNS, as discussed above. One of the primarytools in MS research is experimental autoimmune encephalomyelitis (EAE),a demyelinating condition in animals which mimics many important aspectsof the clinical and pathological features of MS (Prineas and McDonald,1997; Gold et al., 2000). The mechanisms in MS which lead to myelindestruction and the demise of oligodendrocytes are currently unknown.Possible candidates are cell-cell contact involving inflammatory cells,and soluble factors such as TNF-α (Akassoglou et al., 1998),metalloproteinases (Liedtle et al., 1998), reactive oxygen species(e.g., O₂ ⁻ and ONOO⁻) (Kolb and Kolb-Bachofen, 1992), andautoantibodies (Genain et al., 1999). One soluble compound inparticular, which is released in large quantities by activatedleukocytes and microglia, has received little attention: glutamate. Inactivated immune cells, glutamate is produced and released by enzymaticbreakdown of glutamine (Piani et al., 1991; Pithon et al., 1997).However, in animals with EAE, glutamate degradation by astroglialglutamine synthase and glutamate dehydrogenase is diminished(Hardin-Pouzet et al., 1997). These findings suggest an increasedextracellular glutamate concentration in and around the infiltrativelesion. This increase in glutamate is potentially disastrous in themammalian CNS. The extracellular concentration of glutamate is tightlycontrolled in the CNS, and the glutamate gradient between extracellularand intracellular space is about 1:1000. If present in largerquantities, extracellular glutamate can cause excitotoxic cell death byoverstimulation of the cellular ionotropic glutamate receptors, the NMDAand the AMPA/Kainate receptors (Rothman and Olney, 1987; McDonald etal., 1998).

[0122] The inventors have also discovered that glutamate excitotoxicitymediated by AMPA/Kainate receptors accounts for a substantial portion ofCNS damage in EAE (Pitt et al., 2000). Blockage of AMPA/Kainatereceptors by NBQX, a prototypical AMPA/Kainate receptor antagonist,significantly ameliorated the course of the disease and reduced loss ofoligodendrocytes and axonal damage. However, it was demonstrated, bothin vitro and in vivo, that NBQX did not overtly affect the activity ofthe immune system (Pitt et al., 2000). Thus, the observed improvementresulted from direct protection against AMPA/Kainate-receptor-mediatedexcitotoxicity, rather than suppression of the immune response.

[0123] An important event downstream of AMPA/Kainate-receptor-mediatedexcitotoxicity is the opening of voltage-sensitive Ca²⁺ channels, withsubsequent excess Ca²⁺ influx, which results in Ca²⁺ overload andeventual excitotoxic damage (Choi, 1988). Since the inventors hadpreviously established a role for AMPA/Kainate receptor-mediatedexcitotoxicity in EAE, they investigated whether blockage ofvoltage-sensitive Ca²⁺ channels might similarly reduce excitotoxicdamage in this animal model of MS. The calcium-channel antagonistamlodipine was selected for this study because of its long half-life invivo (>30 h), and the beneficial effect on excitotoxic damage which itwas shown to have in an in vitro model for excitotoxicity (Mason et al.,1999).

[0124] As the results of the present investigation show, 30 μg/day ofamlodipine (in 200 μl of phosphate buffered saline [PBS]) significantly(p<0.01) ameliorated clinical impairment in the acute phase of theadoptive-transfer model of EAE. Mice (n=8) were treated from Day 5post-immunization. The control group (n=8) received one dailysubcutaneous injection of 200 μl of the vehicle PBS. The difference inclinical score was significant at Day 4 after onset of the disease, andcontinued to increase until the time of sampling (FIG. 1). The CNSs oftwo representative animals of each group were taken and examined as 1-μmepoxy sections stained with toluidine blue. The animals differedconsiderably in their clinical scoring, with an average score of 1.3(amlodipine group) and 2.8 (control group). However, the examination ofsections from the entire neuraxis (10 levels) showed a similar degree ofinflammation and demyelination in both groups. This indicated thatamlodipine does not modulate the inflammatory process itself—a resultsimilar to that which was found with NBQX treatment.

[0125] In conclusion, the results show that amlodipine significantly(p<0.01) ameliorated the clinical impairment in acute EAE. However,examination of the neuraxis in both vehicle- and amlodipine-treatedanimals showed similar degrees of inflammation, indicating thatamlodipine does not affect inflammation. The reduction in clinicalimpairment is, therefore, most likely due to amlodipine's protecting CNScells against glutamate excitotoxicity via blockage of voltage-sensitiveCa²⁺ channels.

EXAMPLE 2 Additional Calcium Channel Blockers Ameliorate Disease in aMouse Model of Multiple Sclerosis

[0126] An abstract describing portions of this work was published in J.Neurochem. 81:50 (2002), and presented at the Meetings of the AmericanSociety of Neurochemistry, Jun. 22-27, 2002.

Example Summary

[0127] Multiple sclerosis (MS) and experimental autoimmuneencephalomyelitis (EAE), an animal model of MS, are inflammatorydemyelinating diseases of the central nervous system. The inflammatoryattacks lead to glial dysfunction and death, axonal damage andneurological deficits. Numerous in vitro studies suggest thatextracellular calcium influx, via voltage-gated calcium channels,contributes to white matter damage in acute spinal cord injury andstroke. We hypothesized that this mechanism is also operative in EAE,and, possibly, MS. In our study, administration of the calcium-channelblockers bepridil and nitrendipine significantly ameliorated EAE inmice, compared with vehicle treated controls. Spinal cord samples showedreduced axonal destruction in bepridil-treated animals in spite ofmonocyte infiltration to the CNS. Direct protection of axons bycalcium-channel blockers is supported by our immunohistochemical findingthat mouse spinal cord axons express subunits of L-type voltage-gatedcalcium-channel. Our data support the hypothesis that calcium influx viavoltage gated calcium channels plays a significant role in thedevelopment of neurological disability and white matter damage in EAEand MS.

[0128] Introduction

[0129] Multiple sclerosis (MS) is an immune-cell mediated inflammatorydemyelinating disease of the central nervous system (CNS). MS andexperimental autoimmune encephalomyelitis (EAE), an animal model of MS(Juhler, 1988), are characterized by disturbed central nerve conductanceleading to motor and sensory impairments. Pathological findings in MS(Ewing and Bernard, 1998) and EAE (Juhler, 1988) include infiltration ofactivated peripheral inflammatory cells into the CNS (Raine et al.,1984), loss of myelin (Lassman, 1999), edema (Levine et al., 1966) andaxonal damage (Kornek et al., 2000).

[0130] Revealing the still elusive mechanisms that lead to pathologicalchanges in MS would facilitate the therapeutic amelioration of whitematter damage, thus improving neural function. Studies on models ofwhite matter injury have suggested that depolarization and ion balancedisturbances, culminating in increased intracellular calciumconcentration, lead to disrupted axonal function (Rosenberg et al.,1999; Stys and Lopachin, 1998). Decreasing extracellular calciumconcentration also protects rat optic nerve from anoxia (Waxman et al.,1993). Protection of spinal cord dorsal columns from anoxia (Li et al.,2000) or optic nerve against ischemic injury (Stys and Lopachin, 1998)was evident when inhibitors of Na⁺/Ca²⁺ exchange were used. Similarly,spinal cord white matter damage was attenuated by a variety of voltagegated calcium channel blockers in models of anoxia (Brown et al., 2001;Imaizumi et al., 1999) and traumatic injury (Agrawal et al., 2000).

[0131] The above findings, together with the fact that calcium channelsare present on cellular elements (Id.; Brown et al., 2001; Liu et al.,1997) that are key targets in autoimmune demyelination, raise thefollowing question: can calcium antagonists be useful in amelioratinginflammatory demyelination?

[0132] Several lines of evidence support the notion that aberrations inionic balance of white matter constituents contribute to thepathophysiology in MS and EAE (Claudio et al., 1990; Wakefield et al.,1994; Wender et al., 2000). Studies in EAE and MS tissue have suggestedthat involvement of mechanisms such as activation of ionotropicglutamate receptors (Matute et al., 1999; Pitt et al., 2000) and sodiumchannels (Rosenberg et al., 1999) lead to excessive calcium influx.

[0133] Experimental

[0134] We tested the hypothesis that increased influx of extracellularcalcium through voltage-gated calcium channels contributes to theneurological impairment and pathological outcome in EAE, and, byinference, MS. For this, we used two different calcium antagonists,bepridil and nitrendipine. Bepridil is a broad-range calcium-channelblocker with a strong inhibitory effect on Na⁺/Ca²⁺ exchange and onsodium channels (Gill et al., 1992). In contrast, thedihydropyridine-type calcium-channel blocker nitrendipine is a ratherselective blocker of L-type voltage-gated calcium channels (Merck Index,1989). We studied the effect of these drugs on disease onset andprogression using passive, i.e. adoptive transfer, EAE (AT-EAE). AT-EAE,unlike directly induced (active) EAE, is induced by the transfer offully antigen-primed, myelin basic protein-targeted T-cells to recipientanimals (Raine et al., 1984). These primed T-cells are clonally expandedin vitro before the transfer, thus bypassing the initial sensitizationphase of the disease and reducing the possible interference of drugtreatment with development of autoimmunity (Glabinski et al., 1997).

[0135] We used female SJL/J mice (Jackson Laboratories, Bar Harbor, ME).They were housed in a light- and temperature-controlled environment inaccordance with NIH and AAALAC guidelines. All experiments wereperformed under an institutionally approved animal protocol. The antigenwas myelin basic protein, a component of myelin, (1 mg/mouse; Sigma, St.Louis, Mo.) dissolved in sterile PBS and emulsified with an equal volumeof incomplete Freund's adjuvant supplemented with Mycobacteriumtuberculosis (55 μg/mouse; Difco, Detroit, Mich.). The emulsion (200μL/mouse) was injected into the flanks of 11 weeks old mice. Lymph nodecells were obtained from draining lymph nodes 10 days later. Lymph nodecells were cultured for 4 days in RPMI-1640 (Gibco-BRL) medium in thepresence of myelin basic protein (75 μg/ml). Cultured lymph node cellswere subsequently injected into the tail vein of syngeneic 6 weeks oldSJL/J mice at a dose of 4×10⁷ cells/mouse. Onset of disease in controlanimals occurred after 8-10 days. In this animal model of MS, the immuneattack manifests in a predictable pattern of ascending paralysis thatcorrelates with the degree of spinal cord inflammation and edema(Simmons et al., 1982). Disability was scored daily as follows: 0—noclinical signs, 1—flaccid tail, 2—abnormal gait, 3—hind limb paralysis,4—complete paralysis, 5—moribund or dead. Spinal cord tissue, harvestedat the peak of disability, was processed for pathological assessmentusing 1 μm epoxy sections. Animals, with scores representative of eachgroup, were perfused with glutaraldehyde and processed as described(Mokhtarian et al., 1984). Epoxy-embedded 1 μm sections were stainedwith toluidine blue, and examined under a light microscope (OlympusIX-70, 40× objective). For immunohistochemistry, we used longitudinalfreshly-frozen 10 μm sections from phosphate-buffered saline perfusednaive animals, subsequently post-fixed with acetone. Sections weredouble-labeled using SMI31 antibody (1:10,000; Sternberger Monoclonals,MD) for axonal NF-H and a polyclonal anti-Voltage-gated calcium channelssubunit α1D (1 μg/ml; Alomone Labs, Jerusalem, Israel). These primaryantibodies were reacted for 1 hour with Texas Red-conjugated anti-mouse(1:500) and biotinylated anti-rabbit (1:800) antibodies, respectively.Biotinylated secondary antibody was visualized by incubating for 1 hourwith streptavidin conjugated Alexafluor 488 (Molecular Probes, Eugene,Oreg.) at a dilution of 1:2000. Images were acquired using a Bio-Rad(Hercules, Calif.) Radiance 2000 confocal with Nikon (Melville, N.Y.)Eclipse 200 microscope using a 60×N.A. 1.4 planpo objective. A Kr/Arlaser excitation at 488 nm and 568 nm and narrow pass filters detectedgreen and red probes, respectively. For statistical comparison oftreatment groups we used ANOVA followed by multiple comparisons usingthe statistical program Prism 3.0 (Graphpad, San Diego, Calif.). Ap-value of less than 0.05 was considered significant.

[0136] Bepridil (3 mg/kg; s.c.), nitrendipine (2.5 mg/kg; s.c.) andvehicle were administered daily, starting on the day of lymph nodecell-transfer (day 0). Bepridil treatment, from day 0, delayed overtdisease onset by more than 5 days compared with vehicle (FIG. 3), andreduced the clinical impairment beginning at day 10 post lymph nodecell-transfer (p.t.). This difference was statistically significant fromday 13 p.t. onward (p<0.05), continuing into both the plateau phase(days 15-18 p.t.) and the remission phase of the disease (days 19-20p.t.). Bepridil reduced daily average disease scores by 1.8, 2.0, 1.45,1.65, 1.60, 1.60, 1.70 and 1.5 points (average—1.66±0.06) on days 13-20p.t., respectively. With nitrendipine treatment starting at day 0,clinical progression was also slower and reduced when compared withvehicle treated animals (FIG. 4). Average disability scores for thenitrendipine group were lower than corresponding vehicle beginning onday 13 p.t., and this difference became significant (p<0.05) from day 15p.t. Nitrendipine ameliorated the clinical impairment by 1.5, 1.7, 1.9,2.25, 1.65 and 1.6 points (average—1.77±0.11) on days 15-20 p.t.,respectively. There was no statistically significant difference indisability scores between bepridil and nitrendipine groups.

[0137] Studies in various EAE models have demonstrated that the timingof pharmacological intervention is a major determinant in theeffectiveness of treatment (Fujimoto et al., 1999; Jung et al., 1996;Niino et al., 2001). Therefore, we tested whether the mechanisms ofdamage and disability will respond to treatment when it is initiatedlater in the course of the disease. When we started treating mice on day5 p.t., instead of on day 0, neither bepridil nor nitrendipine altereddisease onset or disability compared with vehicle control animals (datanot shown). In contrast, bepridil administration, initiated on day 3p.t. (n=14), reduced neurological impairment significantly (p<0.05) fromday 13 p.t. to day 16 p.t. (the day tissue was harvested), compared withvehicle controls (n=15) (FIG. 2). The difference in average diseasescores between vehicle and bepridil groups was 1.07, 1.20, 0.99 and 1.18points (average—1.11±0.05) on days 13-16 p.t., respectively. In bothtreatment schedules (starting on day 0 or on day 3 p.t.), the differencebetween vehicle control and bepridil treatment became significant on day13 p.t. This difference was larger when treatment started on day 0rather than on day 3 p.t. (difference in average disease scores1.66±0.06 vs. 1.11±0.05, respectively) (FIG. 3). These data suggest adecreasing efficacy for calcium antagonists, as the disease progresses,with the inflammatory attack reaching a ‘point of no return’ between day3 and 5 p.t. These data are in accordance with Morrissey et al. (1996)who reported that, following myelin basic protein-primed T-cellsadministration to rats, the major increase in immune cell countsoccurred between days 3 and 5 p.t. However, in our study, when bepridilwas given 3 days p.t., it did not completely prevent monocyteinfiltration into the CNS, and myclin and axonal damage adjacent to theinfiltrating cells was evident (FIG. 5). Therefore, it seems unlikelythat the ameliorative effect of calcium-channel blockers was due tointerference with immune cell recruitment, although it cannot be ruledout.

[0138] Another possible mechanism for calcium-channel blockeramelioration of AT-EAE is by direct protection of white matterconstituents. In our initial histological data (FIG. 5), axonal loss wasmore pronounced in spinal cords taken from vehicle vs. frombepridil-treated animals. Since the common mechanism for bepridil andnitrendipine is blockade of L-type voltage-gated calcium channels, weused immunohistochemistry to determine whether L-channels are expressedin SJL/J mouse spinal cord white matter. We found abundant overlap ofα1D labeling with axons (data not shown). Control sections for whichanti-α1D antibody was pre-adsorbed with the peptide used to raise theantibody, showed no immunoreactivity to the α1D antibody, confirming thespecificity of the labeling. Our finding is in agreement with Brown etal. (2001) who detected L-channel subunits in rat optic nerve axons.Whether these axonal L-channel subunits are found in the axolemma andform functional channels is still to be determined.

[0139] In summary, two structurally and functionally different calciumantagonists, bepridil and nitrendipine, had comparable beneficialeffects on AT-EAE in mice. They delayed the onset of disability andsignificantly reduced the maximum neurological impairment in this animalmodel of MS. Both drugs, although different in many other respects,block calcium influx through L-type calcium channels. Therefore, it islikely that this mechanism plays a significant role in the tissue damageand the development of neurological disability in inflammatorydemyelinating conditions such as EAE and MS. Our data also supports thenotion that axons may be a direct target for calcium influx viavoltage-gated calcium channels in EAE and MS (Komek et al., 2001).Modulation of calcium influx, either by direct blockade of calciumchannels or by preventing upstream excessive depolarization, may be oftherapeutic value in MS.

[0140] In view of the above, it will be seen that the several advantagesof the invention are achieved and other advantages attained.

[0141] As various changes could be made in the above methods andcompositions without departing from the scope of the invention, it isintended that all matter contained in the above description and shown inthe accompanying drawings shall be interpreted as illustrative and notin a limiting sense.

[0142] All references cited in this specification are herebyincorporated by reference. The discussion of the references herein isintended merely to summarize the assertions made by the authors and noadmission is made that any reference constitutes prior art. Applicantsreserve the right to challenge the accuracy and pertinence of the citedreferences.

What is claimed is:
 1. A method for treating a demyelinating conditionin a subject in need of treatment, the method comprising administeringto the subject an amount of a formulation effective to treat thedemyelinating condition in the subject, the formulation selected fromthe group consisting of a calcium-channel blocker; a combination of acalcium-channel blocker and a glutamate inhibitor; a combination of acalcium-channel blocker and a hypertensive agent; and a combination of acalcium-channel blocker, a glutamate inhibitor and a hypertensive agent.2. The method of claim 1, wherein the demyelinating condition isselected from the group consisting of acute disseminatedencephalomyelitis, acute transverse myelitis, acute viral encephalitis,adrenoleukodystrophy, adrenomyeloneuropathy, AIDS-vacuolar myclopathy,experimental autoimmune encephalomyelitis, experimental autoimmuneneuritis, HTLV-associated myelopathy, Leber's hereditary optic atrophy,multiple sclerosis, progressive multifocal leukoencephalopathy, subacutesclerosing panencephalitis and tropical spastic paraparesis.
 3. Themethod of claim 1, wherein the demyclinating condition is multiplesclerosis.
 4. The method of claim 1, wherein the formulation is acalcium-channel blocker.
 5. The method of claim 4, wherein thecalcium-channel blocker is selected from the group consisting ofamlodipine, bepridil, nitrendipine, diltiazem, felodipine, flunarizine,isradipine, mibefradil, nicardipine, nifedipine, nimodipine,nisoldipine, nivaldipine and verapamil.
 6. The method of claim 4,wherein the calcium-channel blocker is selected from the groupconsisting of amlodipine, bepridil and nitrendipine.
 7. The method ofclaim 4, wherein the calcium-channel blocker is bepridil.
 8. The methodof claim 4, wherein the calcium-channel blocker is nitrendipine.
 9. Themethod of claim 1, wherein the formulation is a combination of acalcium-channel blocker and a glutamate inhibitor.
 10. The method ofclaim 9, wherein the glutamate inhibitor is selected from the groupconsisting of carbidopa, levodopa, sodium channel blockers and NBQX. 11.The method of claim 1, wherein the formulation is a combination of acalcium-channel blocker and a hypertensive agent.
 12. The method ofclaim 11, wherein the hypertensive agent is selected from the groupconsisting of phenylephrine and sodium chloride.
 13. The method of claim1, wherein the agent is administered orally.
 14. The method of claim 1,wherein the agent is administered parenterally.
 15. The method of claim1, wherein the agent is administered by an osmotic mini-pump.
 16. Apharmaceutical composition comprising a calcium-channel blocker, aglutamate inhibitor, and a pharmaceutically-acceptable carrier.
 17. Thepharmaceutical composition of claim 16, wherein the calcium-channelblocker is selected from the group consisting of amlodipine, bepridiland nitrendipine.
 18. The pharmaceutical composition of claim 16,wherein the calcium channel blocker is bepridil.
 19. The pharmaceuticalcomposition of claim 16, wherein the calcium channel blocker isnitrendipine.
 20. A pharmaceutical composition comprising acalcium-channel blocker and a hypertensive agent.